Rare copy number variations in congenital heart disease patients identify unique genes in left-right patterning Khalid A. Fakhroa,b, Murim Choia,b, Stephanie M. Warec, John W. Belmontd, Jeffrey A. Towbinc, Richard P. Liftona,b,1, Mustafa K. Khokhaa,e,1, and Martina Bruecknera,e,1 aDepartment of Genetics, bThe Howard Hughes Medical Institute, and eDepartment of Pediatrics, Yale University School of Medicine, New Haven, CT 06520; cDepartment of Pediatrics, Cincinnati Children’s Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, OH 45229; and dDepartment of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030 Contributed by Richard P. Lifton, January 3, 2011 (sent for review September 21, 2010) Dominant human genetic diseases that impair reproductive fitness embryos; a network of genes involved in the formation and and have high locus heterogeneity constitute a problem for gene function of the ciliated LR organizer that is conserved across all discovery because the usual criterion of finding more mutations vertebrates has been described; however mutations in identified in specific genes than expected by chance may require extremely genes account for less than 10% of affected Htx subjects (9–18). large populations. Heterotaxy (Htx), a congenital heart disease re- A major limitation in identifying causative genes in Htx is the sulting from abnormalities in left-right (LR) body patterning, has paucity of families segregating highly penetrant alleles, and the features suggesting that many cases fall into this category. In this high locus heterogeneity, which has limited the ability to map setting, appropriate model systems may provide a means to sup- disease loci. Because of the marked impairment in reproductive fi port implication of speci c genes. By high-resolution genotyping of fitness, some fraction of Htx could be caused by very rare, highly 262 Htx subjects and 991 controls, we identify a twofold excess of penetrant, dominant mutations. subjects with rare genic copy number variations in Htx (14.5% vs. − Although such mutations have historically been difficult to 7.4%, P = 1.5 × 10 4). Although 7 of 45 Htx copy number variations identify, recent advances have improved the ability to detect were large chromosomal abnormalities, 38 smaller copy number MEDICAL SCIENCES variations altered a total of 61 genes, 22 of which had Xenopus these. For example, the use of quantitative interrogation of dense orthologs. In situ hybridization identified 7 of these 22 genes with sets of SNPs has dramatically improved the ability to detect small fi expression in the ciliated LR organizer (gastrocoel roof plate), a copy number variants (CNVs) (19, 20). The signi cance of such fi marked enrichment compared with 40 of 845 previously studied rare mutations can be dif cult to establish in the setting of high genes (sevenfold enrichment, P < 10−6). Morpholino knockdown locus heterogeneity, as is the case for Htx, where discovering a in Xenopus of Htx candidates demonstrated that five (NEK2, second hit in the same gene in a small cohort is unlikely. Alter- ROCK2, TGFBR2, GALNT11, and NUP188) strongly disrupted both natively, their significance can potentially be assessed in high- morphological LR development and expression of pitx2, a molecular throughput model systems. We have used Xenopus tropicalis, marker of LR patterning. These effects were specific, because 0 of motivated by a conserved mechanism of LR development and 13 control genes from rare Htx or control copy number variations prior use of this animal model system for robust cardiac and gut- produced significant LR abnormalities (P = 0.001). These findings looping assays. Moreover, Xenopus cardiac morphology is more identify genes not previously implicated in LR patterning. similar to human than fish-human similarity (e.g., the presence of atrial septation), and its relatively compact diploid genome (1.5 cardiac development | Xenopus tropicalis | embryo Gb) retains substantial synteny to human, simplifying the identi- fication of orthologous genes (21–23). Additionally, the ability to ongenital heart disease (CHD) is the most common major produce large numbers of embryos and the absence of recent Cbirth defect, affecting ∼1% of live births, yet the cause of genome duplications facilitates screening by morpholino (MO) these lesions remains elusive. Although there is extensive evi- knockdown technology (23, 24). dence from epidemiologic, twin, and animal model studies sup- porting strong genetic contributions to CHD, only a small fraction Results of disease risk has been explained at the molecular level (1). Rare CNVs Are Overrepresented in Htx. We genotyped samples from Heterotaxy (Htx) is a severe form of CHD (2), in which normal 262 subjects with Htx, defined as any arrangement of organs left-right (LR) asymmetry is not properly established, leading to across the LR axis differing from complete situs solitus or malformation of any organ that is asymmetric along the LR axis complete situs inversus (Fig.1 and Table S1). Because there is (Fig. 1). During embryonic development, cardiac precursor cells evidence that human mutations in known LR patterning genes form a symmetric heart tube that undergoes rightward looping to can cause isolated transposition of the great arteries, we included form the geometric framework for the normal four-chambered patients with isolated transposition of the great arteries in our heart. Defects in looping result in a spectrum of complex CHD cohort (17, 25). in ∼90% of Htx patients (3). CHD associated with Htx still has relatively poor survival, despite surgical management. Studies in model systems have established a remarkably well- Author contributions: R.P.L., M.K.K., and M.B. designed research; K.A.F., M.C., M.K.K., and conserved genetic program governing patterning of the verte- M.B. performed research; S.M.W., J.W.B., and J.A.T. contributed new reagents; K.A.F., M.C., brate LR axis (4–6) and cardiac development. LR asymmetry is R.P.L., M.K.K., and M.B. analyzed data; and K.A.F., R.P.L., M.K.K., and M.B. wrote the paper. established during gastrulation at the node (LR organizer) via The authors declare no conflict of interest. dynein-driven, directional beating of cilia. Cilia beating results in Freely available online through the PNAS open access option. fl fl fl leftward ow of extraembryonic uid (nodal ow), which induces 1To whom correspondence may be addressed. E-mail: [email protected], nodal signaling and Pitx2 expression in the left lateral-plate [email protected], or [email protected]. mesoderm (7, 8). Abnormal LR development leads to a striking This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. and specific molecular and anatomic phenotype in all vertebrate 1073/pnas.1019645108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1019645108 PNAS Early Edition | 1of6 Downloaded by guest on October 2, 2021 and duplications that either encompassed an entire coding region or which produced an internal exon duplication. These genic CNVs were annotated for novelty and excluded from further consideration if they were found to have 5% or more overlap with any CNVs, either in the Database of Genomic Variants (http:// projects.tcag.ca/variation/) (27) or in a set of 3,000 control subjects not known to have CHD. We assessed specificity by attempting to confirm the 17 smallest novel CNVs from this set (CNVs en- compassing 19 or fewer SNPs) by quantitative PCR; this set in- cluded seven deletions and 10 duplications. All but a single 10-SNP duplication were confirmed (Methods). fi Fig. 1. Anatomy in human heterotaxy. Situs solitus (SS). The cardiac apex is We identi ed 45 previously unrecorded genic CNVs in 39 oriented leftward, the right lung is trilobed, the left bilobed, the liver is on different subjects (Fig. 2 and Table S2). These CNVs included the right, and the stomach and spleen are on the left. Right atrial isomerism 16 heterozygous genic deletions, 25 complete duplications of at (RAI). Both lungs are trilobed, the liver is midline, and there is asplenia. least one gene, and four internal genic duplications. These CNVs Orientation of the cardiac apex is random, and complex CHD is found were in two size distributions: 38 were relatively small events, in >90% of affected patients. Left atrial isomerism (LAI). Both lungs are affecting one to five genes (27–1,488 kb, mean 2.1 genes per bilobed, the liver is midline, and there are multiple spleens. Orientation of CNV), and seven were larger chromosomal abnormalities, each the cardiac apex is random, and complex CHD is found in 80 to 90% of af- affecting > 90 genes (6–25 Mb, mean >250 genes per CNV). fected patients. Situs inversus (SI). Exact mirror-image of SS: the cardiac apex is rightward, there is a bilobed right lung and a trilobed left lung, the liver is Many more Htx cases than controls had rare genic CNVs [38 of 262 Htx subjects (14.5%) versus 73 of 991 controls (7.4%), P = on the left, and the stomach and spleen are on the right. − 1.5 × 10 4, ratio 2.0:1], consistent with CNVs playing a significant role in Htx development in some patients. To identify CNVs from SNP genotype intensities, we used We focused further evaluation on the 38 CNVs of smaller size, a likelihood-ratio based algorithm (26). In brief, quantitative anticipating that these may identify single genes with large effect intensity values of SNPs previously known to be present at 0, 1, 2, on Htx risk. None of the 61 genes altered by these CNVs had or 3 copies were used to determine the mean and SD of SNP been previously implicated in human Htx or any model of LR intensities for each class.